Oxidation of cytosolic NADH by the malate-aspartate shuttle in HuH13 human hepatoma cells.

1. The reoxidation of cytosolic NADH was studied in a line of human hepatoma cells (HuH13) whose mitochondria preferentially utilized glutamine for ATP formation. 2. The tumor cells showed mitochondrial reoxidation of NADH, as evidenced by the accumulation of pyruvate, when incubated aerobically with L-lactate. The involvement of the respiratory chain was demonstrated by the addition of specific inhibitors. 3. Glutamine oxidation proceeded in the tumor mitochondria exclusively via a pathway involving transamination. Malate stimulated aspartate production from glutamine. 4. When the tumor cells were cultured in Eagle's medium with aminooxyacetate or in the absence of glutamine, a marked reduction in the cellular NAD/NADH ratio was observed. 5. These results indicate that the malate-aspartate shuttle was functioning in the tumor cells.     

Iron regulation of transferrin synthesis in the human hepatoma cell line HepG2

In human beings, serum transferrin levels increase during iron deficiency and decrease with iron overload. Yet, whether or not iron levels actually affect the synthesis of transferrin in human liver cells is not known. In previous studies, iron was shown to suppress the expression of chimeric human transferrin genes in livers of transgenic mice. The goal of this study was to determine if iron suppresses intact endogenous human transferrin synthesis by testing the effects of changes in iron levels on synthesis of transferrin in a human hepatoma cell line HepG2. In HepG2 cells, normalized(35)S-metabolically labeled transferrin synthesis was consistently less following iron treatment with hemin or ferric citrate, than following treatment with an iron-chelator deferroxamine. Thus, this study provides new evidence that iron can regulate synthesis of intact endogenous human transferrin.     

Differential regulation of the JNK/AP-1 pathway by S-adenosylmethionine and methylthioadenosine in primary rat hepatocytes versus HuH7 hepatoma cells

S-adenosylmethionine (AdoMet) and 5'-methylthioadenosine (MTA) exert a protective action on apoptosis induced by okadaic acid in primary rat hepatocytes but not in human transformed HuH7 cells. In the present work, we analyzed the role played by the JNK/activator protein (AP)-1 pathway in this differential effect. Okadaic acid induced the phosphorylation of JNK and c-Jun and the binding activity of AP-1 in primary hepatocytes, and pretreatment with either AdoMet or MTA prevented those effects. In HuH7 cells, pretreatment with either AdoMet or MTA did not affect JNK and c-Jun activation or AP-1 binding induced by okadaic acid. In both cell types, p38 was activated by okadaic acid, but neither AdoMet nor MTA presented a significant effect on its activity. Therefore, the differential effect of both AdoMet and MTA on the JNK/AP-1 pathway could explain their antiapoptotic effect on primary hepatocytes and the lack of protection they show against okadaic acid-induced apoptosis in hepatoma cells.     

Cellular distribution and hormonal regulation of h-SBP in human hepatoma cells

The cellular distribution of human Sex Steroid Binding Plasma Protein (h-SBP) was studied in human cells and tissues by indirect immunofluorescence. h-SBP was detected in the cytoplasm of hepatocytes, of prostate and epididymis epithelial cells and in endometrium. Sexual and non-sexual skin, intestine epithelium, striated muscle and some rodent organs were not labelled. The intracellular localization of h-SBP indicate that h-SBP could be taken up from the extracellular compartment or synthesized in situ in sex steroid target organs, where it may play a role in hormone uptake. The hormonal regulation of h-SBP secretion by a human hepatoma cell line, H5A, showed that tri-iodothyronine was more potent than estradiol or tamoxifen, which acted as estrogen agonist, in increasing secreted h-SBP and the combined effect of both thyroid and estrogen hormones resulted in an additive stimulation of h-SBP secretion. As shown by Northern blot analysis, oligonucleotides synthesized from the known sequence of h-SBP hybridized with a RNA of approximately 2 kb which was more represented in H5A cells than in normal human liver, and was increased 2-3 times after hormonal stimulation of the cells. The presence of a poly(A+)RNA coding for h-SBP in the human liver indicated the hepatic synthesis of this protein.     

Transcriptional regulation of alpha-fetoprotein expression by dexamethasone in human hepatoma cells

The level of alpha-fetoprotein (AFP) mRNA in HuH-7 human hepatoma cells is elevated by the addition of dexamethasone to the culture medium. To locate the DNA region involved in hormonal regulation of the AFP gene, we constructed recombinant plasmids in which various lengths of the 5'-flanking sequence of the human AFP gene were fused to the CAT gene. Various cell lines were transfected with the recombinant plasmids, incubated with or without 3 x 10(-6) M dexamethasone, and then assayed for chloramphenicol acetyltransferase expression. In hepatoma cells that produce AFP, the dexamethasone treatment resulted in the stimulated chloramphenicol acetyltransferase expression when the transfected plasmids contained 169 base pairs (bp) or longer AFP 5'-flanking sequence. No dexamethasone effect was observed when the 5'-flanking sequence was less than 98 bp long. The dexamethasone stimulation was effectively suppressed by the glucocorticoid antagonist RU486, indicating that this effect is mediated by glucocorticoid receptors. The 71-bp region between positions -169 and -98 contains a nucleotide stretch which is similar to the consensus sequence of the glucocorticoid responsive element (GRE). Partial alterations of this sequence resulted in decreased dexamethasone response. The GRE-containing region stimulated heterologous (SV40) promoter activity in response to dexamethasone treatment in an orientation- and position-independent manner. The GRE and the upstream AFP enhancer function independently from each other.     

Depletion of TM6SF2 disturbs membrane lipid composition and dynamics in HuH7 hepatoma cells

A polymorphism of TM6SF2 associates with hepatic lipid accumulation and reduction of triacylglycerol (TAG) secretion, but the function of the encoded protein has remained enigmatic. We studied the effect of stable TM6SF2 knock-down on the lipid content and composition, mitochondrial fatty acid oxidation and organelle structure of HuH7 hepatoma cells. Knock-down of TM6SF2 resulted in intracellular accumulation of TAGs, cholesterol esters, phosphatidylcholine (PC) and phosphatidylethanolamine. In all of these lipid classes, polyunsaturated lipid species were significantly reduced while saturated and monounsaturated species increased their proportions. The PCs encountered relative and absolute arachidonic acid (AA, 20:4n-6) depletion, and AA was also reduced in the total cellular fatty acid pool. Synthesis and turnover of the hepatocellular glycerolipids was enhanced. The TM6SF2 knock-down cells secreted lipoprotein-like particles with a smaller diameter than in the controls, and more lysosome/endosome structures appeared in the knock-down cells. The mitochondrial capacity for palmitate oxidation was significantly reduced. These observations provide novel clues to TM6SF2 function and raise altered mebrane lipid composition and dynamics among the mechanism(s) by which the protein deficiency disturbs hepatic TAG secretion.     

Glucose regulation of acetyl-CoA carboxylase in hepatoma and islet cells.

The regulation of acetyl-CoA carboxylase (ACC) by glucose and other fuel molecules has been examined in Fao Reuber hepatoma cells and Syrian hamster insulin tumor (HIT) cells in order to determine whether lipogenic substrates acutely alter ACC activity and to examine the mechanism of such regulation. In Fao cells, preincubated in simple medium without substrates, glucose addition results in a rapid activation of ACC. This effect, mimicked by other fuels such as lactate, is characterized by an increase in enzyme Vmax and a decrease in the activation constant for citrate. Several lines of evidence indicate that this activation of ACC is due to enzyme dephosphorylation, including the kinetic changes observed, the persistence of enzyme activation through ACC isolation, the necessity of inclusion of sodium fluoride/EDTA in the cell lysis buffer for preservation of the glucose-induced change, and the direct demonstration of diminished 32P-labeling of ACC after glucose exposure. Identical effects of glucose are also observed in HIT cells, although the ACC activation is smaller in magnitude and less sensitive than that observed in Fao cells. Other insulin secretagogues such as glutamine, lactate, and isobutylmethylxanthine are also found to activate HIT ACC. Others have suggested that glucose-induced changes in malonyl-CoA in beta-cells may be linked to glucose-induced insulin secretion. However, studies conducted in late passage HIT cells, which fail to secrete insulin in response to glucose stimulation, reveal the same glucose-induced activation seen in early passages, secretion-competent HIT cells, suggesting that glucose-induced ACC activation is not by itself sufficient to provoke insulin secretion. Taken together, these findings indicate that glucose and other fuel molecules can play a major role in the rapid regulation of the fatty acid synthesis pathway. The activation of fatty acid synthesis by substrate-induced ACC dephosphorylation insures ultimate fuel storage of glucose-derived carbon as fatty acid, while substrate-induced increases in the ACC product, malonyl CoA, would serve to simultaneously limit the rate of fatty acid oxidation through its allosteric regulation of carnitine palmitoyltransferase I.     

IL-1 receptor antagonist regulation of acute phase protein synthesis in human hepatoma cells.

The hepatoma cell line HuH-7 has recently been shown to synthesize serum amyloid A (SAA) in response to IL-1. IL-1 receptor antagonist (IL-1Ra) was able to completely inhibit the response of SAA to IL-1 but not the increase seen in response to IL-6. IL-1Ra was equally effective at inhibiting IL-1 alpha or IL-1 beta. At a 10-fold molar excess of IL-1Ra over IL-1 there was complete inhibition of the SAA response. Removal of IL-1 at 24 h rapidly reduced the SAA secreted over the next 24 h. Addition of IL-1Ra to the cells at this time was as effective as removal of IL-1 at inhibiting the subsequent secretion of SAA. IL-1Ra was less effective at inhibition of IL-1-induced haptoglobin secretion. We would conclude that IL-1Ra may play an important role in the regulation of acute phase protein synthesis.     

Inducible glutamate transport in Mycobacteria and its relation to glutamate oxidation

Washed-cell preparations of Mycobacterium tuberculosis strain H37Ra and M. smegmatis 607 grown in Sauton's medium demonstrated a lag in glutamate oxidation. Washed-cell preparations of M. fortuitum and M. phlei oxidized glutamate immediately and in a linear fashion. Glutamate was oxidized without a lag by washed cells of M. tuberculosis H37Ra and M. smegmatis 607 harvested from a modified medium containing glutamate. Chloramphenicol inhibited the oxidation of glutamate by washed cells grown in the absence of glutamate. These findings suggested the induction of either an enzyme system for glutamate oxidation or a glutamate transport system. The activity of glutamic dehydrogenase was not significantly greater in extracts prepared from cells grown with glutamate. However, the initial rate of glutamate uptake by induced cells was three to four times higher than in noninduced cells. The induction of the glutamate transport system in M. tuberculosis H37Ra and M. smegmatis 607 was shown to parallel the induction of glutamate oxidation. After a 60-min lag, the inducible glutamate transport system appeared. Chloramphenicol prevented the induction of glutamate uptake, although the antibiotic had no effect on glutamate uptake by previously induced cells. Some of the properties of this glutamate uptake system are described.     

Insulin receptor regulation in minimal deviation hepatoma cell line

Treatment of H4 hepatoma cells with the lectin wheat germ agglutinin (WGA) in the concentration range of 10-25 micrograms/ml increased 125I-insulin binding fivefold as compared to control binding in untreated cells. The increased insulin binding was rapid, readily reversible, and correlated with a 10-fold increase in the binding affinity of the receptor for insulin. Kinetic studies indicate that this increased affinity resulted from a decrease in the dissociation rate. The effect was specifically mediated by the lectin since it was reversed by simultaneous incubation with the monosaccharide N-acetyl-D-glucosamine (50 mM) or the disaccharide N,N'-diacetylchitobiose (1 mM). The WGA-mediated increase in insulin binding was not caused by inhibited insulin degradation. While WGA (5 micrograms/ml) mimicked insulin to induce stimulated uptake of [3H]aminoisobutyrate, the lectin failed to enhance the biological sensitivity of H4 hepatoma cells to insulin. At higher concentrations of WGA (125 micrograms/ml), interference with the insulin-mediated response was observed. Trypsin treatment of H4 hepatoma cells prior to measuring binding of 125I-insulin in the presence of increasing concentrations of native insulin, led to a leftward shift of the competition curve, indicating an increased affinity of the receptor. No further increase was observed when the trypsin-treated cells were subsequently exposed to WGA. These results suggest that trypsin treatment and WGA exposure may increase the affinity of the receptor by a similar mechanism. The results are consistent with the concept that WGA and trypsin decrease interaction between insulin binding and receptor affinity regulating components in the plasma membrane, leading to an increase in the affinity of the receptor for insulin.     

The human GLUD2 glutamate dehydrogenase and its regulation in health and disease

Whereas glutamate dehydrogenase in most mammals (hGDH1 in the human) is encoded by a single functional GLUD1 gene expressed widely, humans and other primates have acquired through retroposition an X-linked GLUD2 gene that encodes a highly homologous isoenzyme (hGDH2) expressed in testis and brain. Using an antibody specific for hGDH2, we showed that hGDH2 is expressed in testicular Sertoli cells and in cerebral cortical astrocytes. Although hGDH1 and hGDH2 have similar catalytic properties, they differ markedly in their regulatory profile. While hGDH1 is potently inhibited by GTP and may be controlled by the need of the cell for ATP, hGDH2 has dissociated its function from GTP and may metabolize glutamate even when the Krebs cycle generates GTP amounts sufficient to inactivate hGDH1. As astrocytes are known to provide neurons with lactate that largely derives from the Krebs cycle via conversion of glutamate to α-ketoglutarate, the selective expression of hGDH2 may facilitate metabolic recycling processes essential for glutamatergic transmission. As there is evidence for deregulation of glutamate metabolism in degenerative neurologic disorders, we sequenced GLUD1 and GLUD2 genes in neurologic patients and found that a rare T1492G variation in GLUD2 that results in substitution of Ala for Ser445 in the regulatory domain of hGDH2 interacted significantly with Parkinson's disease (PD) onset. Thus, in two independent Greek and one North American PD cohorts, Ser445Ala hemizygous males, but not heterozygous females, developed PD 6-13 years earlier than subjects with other genotypes. The Ala445-hGDH2 variant shows enhanced catalytic activity that is resistant to modulation by GTP, but sensitive to inhibition by estrogens. These observations are thought to suggest that enhanced glutamate oxidation by the Ala445-hGDH2 variant accelerates nigral cell degeneration in hemizygous males and that inhibition of the overactive enzyme by estrogens protects heterozygous females. We then evaluated the interaction of estrogens and neuroleptic agents (haloperidol and perphenazine) with the wild-type hGDH1 and hGDH2 and found that both inhibited hGDH2 more potently than hGDH1 and that the evolutionary Arg443Ser substitution was largely responsible for this sensitivity. Hence, the properties acquired by hGDH2 during its evolution have made the enzyme a selective target for neuroactive steroids and drugs, providing new means for therapeutic interventions in disorders linked to deregulation of this enzyme.     

Effect of gossypol on intracellular Ca2+ regulation in human hepatoma cells

Gossypol is a natural toxicant present in cottonseeds, and is hepatotoxic to animals and human. The effect of gossypol on cytosolic free Ca2+ levels ([Ca2+]i) in HA22/VGH human hepatocytes was explored using fura-2 as a fluorescent Ca2+ indicator. Gossypol increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 2 microM. The Ca2+ signal was reduced by removing extracellular Ca2+ or by 10 microM La3+, but was not affected by nifedipine, verapamil or diltiazem. Pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) to deplete the endoplasmic reticulum Ca2+ partly reduced 10 microM gossypol-induced Ca2+ release; and conversely pretreatment with gossypol abolished thapsigargin-induced Ca2+ release. The Ca2+ release induced by 10 microM gossypol was not changed by inhibiting phospholipase C with 2 microM U73122 or by depleting ryanodine-sensitive Ca2+ stores with 50 microM ryanodine. Together, the results suggest that in human hepatocytes, gossypol induced a [Ca2+]i increase by causing store Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent manner, and by inducing Ca2+ influx.     

Hormonal regulation of carbamoyl-phosphate synthetase I synthesis in primary cultured hepatocytes and Reuber hepatoma H-35. Defective regulation in hepatoma cells

Regulation of carbamoyl-phosphate synthetase I (CPS) synthesis by various hormones was compared in primary cultured hepatocytes from adult rat and in Reuber hepatoma H-35 by pulse labeling of the cells with [35S]methionine. CPS synthesis in hepatocytes was stimulated 8-fold and 5-fold by dexamethasone and glucagon respectively. CPS synthesis in hepatocytes was synergically (about 50-fold) stimulated by a combination of dexamethasone and glucagon. Less synergic stimulation was observed by combining dexamethasone with N6, O2'-dibutyryladenosine 3',5'-monophosphate (dibutyryl-cAMP) or with isoproterenol. The basal level of CPS synthesis in hepatoma cells was higher than that in hepatocytes. CPS synthesis in hepatoma cells was stimulated by dexamethasone and dibutyryl-cAMP but the extent was only 3-fold and 1.8-fold respectively. The synergic effect of combination of dexamethasone and dibutyryl-cAMP was not observed in hepatoma cells. Neither glucagon nor isoproterenol exhibited an appreciable effect on CPS synthesis in hepatoma cells. Insulin and epinephrine suppressed CPS synthesis both in hepatocytes and hepatoma cells. The effect of epinephrine was indicated to be through alpha-adrenergic receptors. The effects of insulin and epinephrine were additive on CPS synthesis both in hepatocytes and hepatoma cells.     

Identification of major proteins in the lipid droplet-enriched fraction isolated from the human hepatocyte cell line HuH7

Recent studies have revealed the presence of intracellular lipid droplets in wide variety of species. In mammalian cells, there exist proteins specifically localize in lipid droplets. However, the protein profile in the droplet remains yet to be clarified. In this study, a fraction enriched with lipid droplets was isolated from a human hepatocyte cell line HuH7 using sucrose density gradient centrifugation, and 17 major proteins in the fraction were identified using nano LC-MS/MS techniques. Adipose differentiation-related protein (ADRP) was the most abundant protein in the fraction. The secondary abundant proteins were identified to be acyl-CoA synthetase 3 (ACS3) and 17beta-hydroxysteroid dehydrogenase 11 (17betaHSD11). Included in the identified proteins were five lipid-metabolizing enzymes as well as two lipid droplet-specific proteins. When HuH7 cell lysate was fractionated by a density gradient, most of 17betaHSD11 was found in the droplet-enriched fraction. In immunocytochemical analysis, 17betaHSD11 showed ring-shaped images which overlapped with those for ADRP. These results suggest that a specific set of proteins is enriched in the lipid droplet-enriched fraction and that 17betaHSD11 localizes specifically in the fraction.     

Expression of haptoglobin receptors in human hepatoma cells.

The uptake of radio-labeled hemoglobin-haptoglobin complex (Hb-Hp) by human hepatoma PLC/PRF/5 and HepG2 cells was investigated in an attempt to characterize the uptake process and intracellular transport. Human hepatoma cells took up Hb-Hp in a receptor-mediated manner. Scatchard analysis of binding revealed that PLC/PRF/5 and HepG2 cells exhibited about 21,000 and 63,000 haptoglobin receptors/cell, with a dissociation constant (Kd) of 8.0 and 17 nM, respectively. Human hepatocytes in primary culture also expressed about 84,000 receptors/cells, with a Kd of 7.4 nM. The hemoglobin-haptoglobin complex was internalized and subsequently the internalized Hb-Hp was slowly degraded in the cells. Preincubation of the cells with Hb-Hp resulted in a decrease in binding of the radioactive Hb-Hp to the cell surface, and was accompanied with an accumulation of intracellular receptors. The uptake of Hb-Hp by the cells was not inhibited by 100 microM chloroquine or by 10 mM methylamine, but was inhibited by 50 microM monodansylcadaverine. Hemoglobin-heme taken up by the cells induced microsomal heme oxygenase. Thus, human hepatoma PLC/PRF/5 and HepG2 cells can take up Hb-Hp by haptoglobin receptor-mediated endocytosis and Hb-Hp probably causes translocation of the haptoglobin receptors from the cell surface to the cell interior where they can be degraded. The internalized heme-moiety of hemoglobin can regulate the expression of heme oxygenase.     

Rapid acidification of endocytic vesicles containing asialoglycoprotein in cells of a human hepatoma line

Acidification of endocytic vesicles has been implicated as a necessary step in various processes including receptor recycling, virus penetration, and the entry of diphtheria toxin into cells. However, there have been few accurate pH measurements in morphologically and biochemically defined endocytic compartments. In this paper, we show that prelysosomal endocytic vesicles in HepG2 human hepatoma cells have an internal pH of approximately 5.4. (We previously reported that similar vesicles in mouse fibroblasts have a pH of 5.0.) The pH values were obtained from the fluorescence excitation profile after internalization of fluorescein labeled asialo-orosomucoid (ASOR). To make fluorescence measurements against the high autofluorescence background, we developed digital image analysis methods for estimating the pH within individual endocytic vesicles or lysosomes. Ultrastructural localization with colloidal gold ASOR demonstrated that the pH measurements were made when ligand was in tubulovesicular structures lacking acid phosphatase activity. Biochemical studies with 125I-ASOR demonstrated that acidification precedes degradation by more than 30 min at 37 degrees C. At 23 degrees C ligand degradation ceases almost entirely, but endocytic vesicle acidification and receptor recycling continue. These results demonstrate that acidification of endocytic vesicles, which causes ligand dissociation, occurs without fusion of endocytic vesicles with lysosomes. Methylamine and monensin raise the pH of endocytic vesicles and cause a ligand-independent loss of receptors. The effects on endocytic vesicle pH are rapidly reversible upon removal of the perturbant, but the effects on cell surface receptors are slowly reversible with methylamine and essentially irreversible with monensin. This suggests that monensin can block receptor recycling at a highly sensitive step beyond the acidification of endocytic vesicles. Taken together with other direct and indirect estimates of endocytic vesicle pH, these studies indicate that endocytic vesicles in many cell types rapidly acidify below pH 5.5, a pH sufficiently acidic to allow receptor-ligand dissociation and the penetration of some toxin chains and enveloped virus nucleocapsids into the cytoplasm.